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Escherichia coli as a Model Organism and Its Application in Biotechnology
By Vargas-Maya Naurú Idalia and Franco Bernardo
Submitted: May 18th 2016Reviewed: December 20th 2016Published: July 12th 2017
Home > Books > Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications
Without a doubt, in the past 20 or so years, we have achieved the power of biology in different ways. In the present, we have many tools for developing novel technologies and applications for organism modifications that ultimately let us know many aspects of organisms' biology and, therefore, apply that knowledge for technological purposes. Of all the model organisms and tools for genetic modification available, Escherichia coli stands out as a model organism and what we would like to call "molecular biologist tool box." In the present chapter, we aim to review our current knowledge regarding genetic modifications and tools for modifying E. coli to generate plasmid vectors, single and multiple gene knockouts, whole genome editing, biosensor generation and applications and synthetic gene circuits and genomes.
bioengineerging
Vargas-Maya Naurú Idalia*
Biology Department, Division of Natural and Exact Sciences. University of Guanajuato, México
Franco Bernardo
*Address all correspondence to: naudalia@hotmail.com
Escherichia coliRecent Advances on Physiology, Pathogenesis and Biotechnological ApplicationsEdited by Amidou Samie
Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications
Edited by Amidou Samie
Who in biology has not heard of Escherichia coli? Known to many as the fundamental model microbe and perhaps model organism, E. coliis the cornerstone of many important findings in molecular biology and other areas of cell physiology. Perhaps even the first chemo-organoheterotroph had a similar mass composition as E. coli, providing the hits necessary to understand the evolution of modern bacteria [1]. Also called the "workhorse" of molecular biology for its fast growing rate in chemically defined media and extensive molecular tools available for different purposes, E. coliis considered the most important model organism of them all. Important findings and Nobel Prizes in biology have been developed in E. coli. For instance, cracking the genetic code [2], unveiling the nature of DNA replication [3], the groundbreaking advances on gene organization and regulation or as we love to call 'the operon' [4, 5], important evidence for the basis of mutations and ultimately to the evolution of organisms [6, 7], and finally, the achievement of a genetically modified organism [8] that skyrocketed several applications of the enormous capacity for manipulating this organism, rendering E. colias a key player in biotechnology (for an excellent review, see Ref. [9]).
The main reasons why E. coliis the organism of choice extends and is not limited to its fast growth in chemically defined media; relative cheap culture media; does not form aggregates; industrial scalability; several molecular tools for manipulation; extensive knowledge of its genetics and genomics; extensive knowledge on its transcriptome, proteome, and metabolome, and several strains are considered biosafety 1, which renders it ideal even for teaching and school demonstrations (for example, Bio-Rad transformation kits, URL: http://www.bio-rad.com/es-mx/category/pglo-plasmid-gfp-kits).
Typical E. coli, a Gram-negative bacillus bears a rod-shaped, measures only about 1 μm long by 0.35 μm wide, although this can vary considerably depending on the strain and its conditions, there are studies regarding different mutations that affect its size and length considerably. A recent paper showed that this bacterium can grow up to 750 μm in length. By random Tn10 insertion, they found this particular mutant has an insertion at genes ybdN and ybdM whose function remains uncertain and another mutation that remains to be identified [10]. Interestingly, this mutant strain gives rise to extremely long cells that are viable (capable of cell division) and retain metabolic activity that can be useful for studies involving intracellular localization or optimize cell-surface contact.
In terms of ecology, E. coliis a facultative aerobe (either respiration takes place in the presence of oxygen or fermentation in its absence), which bears a sensor for oxygen presence (redox state in the quinone pool) and can activate or repress the required metabolic enzymes, depending on oxygen levels [11–13]. Also, E. coliand other Enterobacteriaceae can be the first organisms to colonize human infant intestine due to this capacity to metabolize oxygen or ferment because this facultative bacteria will consume the remaining oxygen in this environment so that other strict anaerobic bacteria are capable of colonizing the intestine rendering the normal microbiota found in humans and also thrive as part of the normal microbiota [14].
Phylogenetically, E. coliis a member of the Enterobacteriaceae and is closely related to pathogens such as Salmonella, Klebsiella, Serratia, and the infamous Yersinia pestis, which causes plague. Although E. coliis mostly harmless, pathogenicity islands have been identified and associated with pathogenesis in E. coliresulting in strains that colonize different tissues [15].
The building blocks of E. coliconsists of about 55% protein, 25% nucleic acids, 9% lipids, 6% cell wall, 2.5% glycogen, and 3% other metabolites [16–18], which for biotechnological applications is important, since carbon flux is often a problematic issue to address in order to generate a novel metabolic pathway or to enhance a current functioning pathway. Also, carbon flux is tightly regulated by sophisticated regulatory networks that require modeling and a basic understanding of the regulatory mechanisms in order to manipulate them and achieve desired goals [19–21].
E. coliis part of the normal microbiota of mammals, rendering the predominant facultative microbe of the gastrointestinal tract and is currently a hot debate on the impact on normal microflora establishment and their role in disease [22].
This organism lacks many interesting features for biotechnology, such as growing at extreme temperatures or pH, the capacity to degrade toxic compounds, pollutants, or difficult to degrade polymers [23]. But as we will see later, this bacterium is capable of doing amazing things and its only limit is our imagination.
E. coliharbors a genome with particular features such as a strikingly organized structure, remnents of many phages, and insertion sequences (IS) and a high transport capacity toward the cytoplasm. In 1997, the complete genome sequence of the K-12 strain was obtained, and a myriad of research was catapulted from that moment on [24]. The complete genome contains a single circular duplex molecule composed of 4,639,221 bp. Regarding its structure, protein-coding regions correspond to 87.8% of the genome, while 0.8% encodes for stable RNAs, and 0.7% consists of noncoding repeats. The remaining 11% encodes for regulatory and other functions. Nevertheless, nearly 34% (1431) proteins are considered orphan or without defined molecular function but in a recent study, it was demonstrated that by homology with distant phylogenetical relationships, they may play a role in defined molecular pathways or processes [25]. From the orphan set in E. coli, at least 446 contain some molecular signature that can assess their molecular role. The fact that such a vast portion of the genome remains uncharacterized for defined molecular pathways or roles paves the way for future basic research that may have a strong impact on applied research and development. For biotechnological applications, growth optimization is also a required feature of genetically modified organisms. For E. coli, it has been recently shown that growth can be manipulated by removing nonessential genomic sequences that also can lead to the design of important biotechnological strains for industrial purposes [26].
The advancement of genomic, transcriptomic, and proteomic technologies has led to the development of several online resources for the analysis of molecular and physiological aspects of E. coli. Some examples are given in Table 1.
Escherichia colidatabases
EcoCyc: Encyclopedia of E. coliGenes and Metabolic Pathways https://ecocyc.org A comprehensive database joining together genomic information with biochemical features of E. coli
PortEco http://www.porteco.org A resource for knowledge and data of the biology of E. coliincluding plasmids, mobile genetic elements, and phages
EcoliWiki http://ecoliwiki.net/ Community-based pages about everything related to the biology of the nonpathogenic E. coli
EcoGen 3.0 http://ecogene.org/ A database dedicated to analyzing and comparing genomic and transcriptomic data
Kegg http://www.genome.jp/kegg-bin/show_organism?org=eco A powerful resource for understanding molecular datasets in different contexts, also easy access to gene information
RegulonDB http://regulondb.ccg.unam.mx Database on transcriptional regulation in E. coliK-12 containing knowledge manually curated from original scientific publications, complemented with high throughput datasets and comprehensive computational predictions
E. coliGenetic Stock Center http://cgsc.biology.yale.edu/ The CGSC Database of E. coligenetic information includes genotypes and reference information for the strains in the CGSC collection. An excellent resource for acquiring information and strains for research
Relevant E. coliresources and databases.
*Source: databases website.
Recently, using the Genome Conformation Capture technique, it was revealed that the linear organization of the genome is also true for the 3D structure, rendering neighboring genes to form small factories that are coregulated or coexpressed and showed a higher probability of forming protein-protein interactions. This organization represents two important aspects of bacterial genomes, first, the compactness in the 3D space, containing pathways in a nonrandom distribution, and second, genes that are closer to each other tend to be coexpressed and form protein-protein interactions favoring the concept of transcription factor even in microbial cells [27].
In the following sections, we will review what we consider the modern tools for genetic engineering E. coli, and the future for this microbe that can be considered the toolbox for molecular biology and may be the answer to many problems that humanity may face in the future.
2. Plasmid and the E. colirevolution
Without a doubt, plasmids are the most important tools not only for the manipulation of E. colibut also the foundation for the genetic engineering of many organisms, cloning and sequencing, generation of mutants, and many applications in molecular biology. In this section, a brief summary of the myriad of plasmids available will be addressed, providing some of the most important features of plasmids and the up-to-date technologies available for plasmid manipulation and application.
Why plasmids are the basis for genetic engineering? By surveying the literature and commercial sources catalogs, there is a myriad of applications for plasmids: cloning, mutagenesis, protein fusion and overexpression, shuttle vectors from bacteria to a diverse range of hosts, among others. Plasmids must be first presented and then we provide some features that are relevant regarding the importance of plasmids in molecular biology and biotechnology.
Plasmids are extrachromosomal molecules that are self-replicative and sometimes provide interesting features to its host. The term was first coined by Joshua Lederberg in 1952 referring to genetic elements in bacteria that remained as an independent molecule from the chromosome at any stage of their replication cycle [28]. The definition was further refined to all the autonomously replicating DNA molecules to avoid including viruses. These molecules are present not only in eubacteria but also are found in Archea and some lower eukaryotic organisms [29]. In nature, many bacteria contain self-replicating DNA molecules that can be harnessed for molecular biology applications. E. coliplasmids were the first ones to be extensively modified for such purposes [30].
In the 1970s, the first generation of cloning plasmids was created, and from that moment on, research in the biological area was enriched with a powerful tool. Plasmids must contain several important features to be used in research: proper size for ease to transform or transfect, selection markers, a replication origin, regulatory elements to control expression, and transcription termination. All features are important when designing a plasmid vector for the desired application, the reader can imagine the goal, and there will always be a way to create the molecular tool for achieving such a goal, and that is possible due to the basic structure of most plasmids used in molecular biology and their modularity [31]. In Table 2, we summarize some of the most important features (modules) that plasmids must have in order to serve for different applications. We point out that sequence composition and structure, copy number, selection marker, and special features such as reporter proteins or regulatory elements are the most important features in a plasmid and can influence the outcome of the desired application.
Escherichia coliplasmids
Type of element
ColE1 Replication origin Generates 15–20 copies of each plasmid molecule. Colicin production. Related to plasmids that confer immunity to phage infections [32]. Found in low copy plasmids such as pBR322 [33]. There are mutations in this replication origin that leads to high copy number plasmids, such as pUC series that can render up to 700 copies per cell [34, 35]
p15A Replication origin Low copy number replication origin, estimated in 18–22 copies per cell [36]. This type of replication origin is often found in pACYC and its derivative vectors
pMB1 Replication origin Versatile replication origin. The original sequence generates 15–20 copies per cell, but a mutant version can lead up to 700 copies per cell [37]. This plasmid contains the EcoRI restriction-modification system
pSC101 Replication origin Five copies per cell [38]
R6K Replication origin 15–20 copies per cell. Requires the π protein from the gene pirfor replication [39]. This origin of replication is functional in diverse bacterial species
Ampr, Kanr, Cmr, Tetr among other Selection markers Elements required for the selection and maintenance of plasmids in bacterial hosts. Here are listed the resistance cassettes for Ampicillin, Kanamycin, Chloramphenicol, and Tetracycline, which are the most common selection markers. For additional markers, RAC database contains the information regarding antibiotic resistance traits and their sequence [40] or iGEM website for sequence modules bearing the proper syntax for synthetic constructs
LacZ, CcdB, Green Fluorescent protein (GFP), etc Additional elements required for positive clone selection, reporter protein fusions among others Plasmids have been modified so that they contain multiple cloning sites with diverse unique restriction sites, counter selection for positive clone selection. Additional elements such as filamentous origins for single-stranded DNA generation for sequencing of high G+C templates or site-directed mutagenesis
Common elements in plasmids used in molecular biology applications.
Additional sources: [31], http://blog.addgene.org/plasmid-101-origin-of-replication. https://www.neb.com/, https://www.thermofisher.com/mx/es/home/brands/invitrogen.html, and http://parts.igem.org/Help:Synthetic_Biology, which contains a repository of parts that can be needed for plasmid construction using synthetic methods.
Some exceptional features of plasmids are they can be used in systems where replication origins (check compatibility first) and selection markers can coexist in the same cell, which can be extremely useful for the coexpression of four different proteins in the same cell (e.g., the four plasmid system developed by Dykxhoorn et al., which are compatible between them [41]); the broad diversity of selection markers and partitioning control elements [30]; cloning capacity [31], which is an important feature for cloning large fragments required for synthetic biology applications or metabolic engineering; reporter proteins useful for selecting positive clones; recombination or assembly technology for easier cloning methods [42, 43], and the ability to be transferred from one host to another like the case of the OriV from RK2 plasmid [44]. We recommend for further information about replication control of plasmids to refer to del Solar et al. [45].
Linear plasmids are common in bacteria (particularly in actinobacteria), but thus far, only N15 plasmid prophage has been isolated from E. coli[46] and is an impediment for generating knockouts using linear DNA (see Section 3). Recently, a linear plasmid was created to clone unstable fragments bearing repetitive sequences without showing size bias during cloning, active promoter sequences, or sequences with A + T content [47].
One of the biggest impediments for plasmid segregation is the insert size. As discussed in Section 5, we are now facing not only the most exciting age of molecular biology but also the most challenging. In order to develop "bugs to the order," i.e., microbes are "trained" to perform specific tasks [48], from simple pathways to synthesize a specific metabolite to complex genetic circuits that can be controlled for novel environmental responses. In all these cases, plasmids play an important role, from generating site-specific integration of chromosomal fusions to large plasmids that can hold complex constructs for further modification. Another challenging area is the generation of strains capable of producing metabolic products required for the pharmaceutical industry, where metabolic pathways and cell metabolism can be a strong impediment for proper synthesis and purification of relevant precursors [49]. E. colirenders an important platform for tackling down impediments for the synthesis of novel compounds.
As part of the information needed for plasmid manipulation, databases and repositories are also relevant for the manipulation and selection of the right plasmid for the applications you want to further exploit. Such examples are given: a powerful plasmid repository is Addgene (https://www.addgene.org/vector-database/) where you can get any plasmid in the repository with minimum fees for shipping and handling. This source is important in many aspects; you can gain knowledge about plasmid sequence, special features, creators, and availability to use since you can acquire them with small fees. Also, Harvard University is currently generating a plasmid repository but still under development (https://plasmid.med.harvard.edu/PLASMID/Home.xhtml). These repositories are an excellent option for accelerating research due to finding already generated constructs useful for ongoing projects. Also, E. coliGenetic Stock Center (Table 1) is a good source of strains and plasmids for different applications. For a small fee, the strains and plasmids can be shipped worldwide and characteristics can be consulted. Also, the American Type Culture Collection (www.atcc.org) is a source for strains with desired characteristics, as well as knowledge on their features. In plasmid biology, strain selection is fundamental for plasmid stability and proper propagation, as well as for the correct function of special features, such as protein expression [50]. For example, DNA methylation is an important impediment in certain applications such as eukaryotic transfection, proper DAM− and DCM− strains for plasmid isolation is required. In protein expression experiments, the repertoire for host selection is big. Most applications require lysogen strains harboring the required RNA polymerase. As stated in Table 2, some plasmids require the product from the pirgene, so careful strain selection must be taken into account so that plasmids replicate efficiently.
Novel methods such as Gibson assembly, Golden Gate assembly, and AQUA (advanced quick assembly) methods [43, 51, 52] have skyrocketed the possibility to assemble any plasmid with the desired characteristics. These methods are based on designed modules that can either be Polymerase Chain Reaction (PCR) amplified or generated as a complete synthetic construct and then assembled in the desired combination either by an enzymatic process (Gibson and Golden gate) or even enzyme-free methods such as AQUA.
Finally, plasmid biology is still under scrutiny, for their involvement in the mobility of traits that are important for human health such as antibiotic resistance, the distribution of pathogenicity islands, and genome evolution. Recently, novel tools for plasmid mining have been developed and uncover from Next Generation Sequencing data that plasmids can be uncovered and analyzed for further characterization [53, 54].
We are still in the process of truly knowing the potential of E. coli, novel tools generated through plasmids in combination with other molecular strategies will lead to new discoveries that will render this organism the basis for important discoveries. In the next section, we will discuss some aspects of gene knockouts and the knowledge we have gained from this versatile organism.
3. Genome modifications to understand E. coli
One of the basic questions in biology is: what is life? Defining life imposes a challenging burden both intellectual and experimental. Many attempts have been done to answer this. E. coliis a model organism that with all the molecular tools available we can get a step closer to provide sufficient information that will lead us to answer the relevance of genomic information and ultimately what is needed to achieve life [55]. In 1997, its genome was fully sequenced, and with all that information (from E. coliand all the organisms that have been sequenced thus far), scientists have aimed high to achieve the knowledge of how many genes are needed for life to be sustained. Molecular genetics have provided many tools for understanding gene structure and function, the most fundamentals are gene knockouts and genome deletions. In this section, we provide aspects that are fundamental for understanding genome structure and function taking our knowledge closer to knowing the minimal core genome of bacterial organisms and the optimization of E. colifor biotechnology.
In E. coli, several tools for genome modification have been developed. Some of the most important methods involve either the generation of deletion mutants by removing specific genes, one outstanding case is the use of the lambda Red system for inhibiting linear DNA degradation and by homologous recombination, the deletion of specific genes using PCR-derived selection marker cassettes with homologous sequences with target gene [56, 57]. Lambda Red-based method have yielded a total of 4288 genes mutated without lethality (Keio collection), 303 genes were unable to be deleted, from which 37 are of unknown function [58]. This experimental evidence has pointed out one very important aspect of genome structure and function. Genome size increase is the result of horizontal gene transfer or DNA fragment retention that somehow is giving some beneficial features to recipient host, apparently an increase in fitness [26]. The function of genes without evident function is still a relevant area of research since many of them provide support for fitness and evolution has preserved them, therefore full genome engineering is far more complicated than previously thought.
Larger genomic editions are needed to understand how far we can delete redundant or nonessential sequences. By using Cre/lox recombination, substantial genomic fragments can be deleted or sequentially removed, rendering the nonessential regions (regardless the genes present) from the genome [59–61].
Studies regarding genome size analyzed through deletions of specific genes or complete genomic regions have led on thinking about the minimal genome. In the case of E. coli, there are several pieces of evidence (reviewed in Ref. [62]) that points out that at least 23% of the genome can be eliminated gaining genomic stability and normal growth. Also, eliminating insertion sequences can enhance the capacity of E. colito synthesize proteins due to the decrease or insertions on plasmids, and strains exhibit normal growth plus increased genome stability [63].
All these methods rely on basic bacterial genetics founded with E. coli, such as transposon-based integration of recombination sequences, λ-recombination of PCR products integrating deletion module cassettes, and the gene-specific knockout methods [62]. Mutations can then be transferred from one strain to the other to generate multiple deletions at once, and other technologies are still limited to either whole genome synthesis with previous knowledge on the structure of the genome.
The most relevant study revealed that genome size has an impact on E. colicell growth, where it is shown that apparently dispensable sequences are needed under restrictive conditions, providing a hint of the still far future of fully functioning cells with all the desired characteristics for biotechnological applications [26]. We envision that genome reduction is a worthy effort, regardless of the method used to generate them. Another important aspect that we have to consider is that all conditions of the mutant strains are exposed to laboratory conditions rendering a behavior close to the ancestor or original strains. Nevertheless, there are also hidden features that must be exploited in order to understand fully the behavior of the genome and the essentiality of genes [62]. Thus far, E. coliremains restricted to the use of classic genetic tools and transposon or plasmid-based techniques. We encourage E. coliresearch community to join efforts to enter the synthetic biology era, toward the generation of a fully synthetic E. coli. Our excitement is based on the following:
After 6 years, in 2016, the first bacteria operating under a "minimal chemically synthesized genome" was created after the first fully functioning synthetic genome [64, 65].
This research we believe has an impact in the following areas. First, both studies settled the basis for whole genome synthetic biology, which will lead to important findings in many research areas. Second, the extensive transposon-based mutagenesis studies on the genome of Mycoplasma mycoidesled to the knowledge of the basis of essential genes or quasi-essential genes that have an important impact on cell fitness. Third, all this knowledge led to the design of a complete chemically synthesized genome with all the basic functions, and we now have the basic information for mining existing genomes to look for core modules in the bacterial genomes and design genomes with specific functions. Taking together all the observations from the synthetic genomes, we envision a bright future for bacterial molecular genetics in many fields of biotechnology, such as the production of molecules for human wellbeing.
E. coliis an extensively studied organism, with all the cumulative data we can ensure that with all this knowledge, we can design tools. In the following section, we comment on the biosensors that are E. coli-based.
4. E. coli-based biosensors: tools for many applications
In biotechnology, biosensors are broadly defined as any device based on biological part, cell, tissue, or protein complex that are linked to a mechanical sensor or analytical receptor that provides a measurable signal proportional to the analyte in the reaction [66, 67]. E. coli-based biosensors using plasmid or chromosomal constructs are useful for the detection of environmental traits or hazards or measuring cellular processes as any standard reporter system [68–70].
In Figure 1, we depict the basic design for whole-cell biosensors and some applications. Plasmid vectors with all the possible modifications can lead to almost endless combinations. For practical applications, there are commercial vectors that can be used for such purposes or as mentioned in the previous sections, plasmid methods are powerful enough for fast and robust biosensor design.
Basic principles for biosensor design. First, the proper design and the experimental creation of reporter strains. With current knowledge, either plasmid creation or whole genome engineering can lead to the creation of a reporter strain. Second, incorporation of such elements into the host cell. Third, sensing module and response measurement. With current reporter proteins and detection technology, it is relatively easy to generate biosensors that can be used in different applications with high sensitivity and selectivity.
The basic design considers the following: copy number, reporter proteins, detection methods, and control elements. The latter is basically the most important feature. As shown in Table 1, the available databases provide enough information for promoter selection and design. Bioinformatic tools can make this process easier [71]. Also, generation and detection of this kind of biosensors are cost-effective and easy to generate and reasonably sensitive [71]. In terms of speed, sample analysis with whole-cell biosensors is fast and cheap in comparison with analytical methods. The sensitivity of analytical methods is higher and more accurate, but biosensors are a good alternative for fast detection of hazards. Also, they can be coupled with the controlled production of metabolites of commercial importance.
In the literature, there are several reports where E. coli-based biosensors have been successful for detecting different traits: oxidants [72, 73], DNA damaging compounds [74], membrane-damaging compounds [75], protein-damaging compounds [76], aromatic compounds [77–79], xenobiotics [80], antibiotic panels using reporter strains without antibiotic selection [81], etc. The only limitation is the available sensor module and the design. The reporter protein is also important. Stability and reproducibility are two important aspects of biosensor design. In our experience, Green Fluorescent protein (GFP) protein is superior to luciferase, especially that we can detect GFP by various methods (we find flow cytometry, fluorometry, and confocal microscopy our top preferences) without cell lysis or substrate mixtures that are time-consuming [82].
With the improvement of DNA synthesis, recoding protein-coding genes for the desired function is expanding the capabilities of transcription factors, and reporter proteins have created novel sensor modules. For example, XilR recoding has led to a sensor that can detect millimolar concentrations of trinitrotoluene and its derivative compounds [83]. By using shuttle vectors, we can generate biosensors that we can transfer from one host to another, which can provide information about differences in physiological responses during the exposure to a given environmental trait.
E. coliplasticity and tools such as BioBrick building (using standardized DNA fragments with compatible ends for fast assembly) can facilitate plasmid and reporter constructs [84, 85]. Correlations of cell growth and physiology with expression patterns from reporter constructs can expand our knowledge of the impact of exposure to the external stimulus on cell physiology. Biosensors based on whole cells are a cheap alternative and can be coupled to portable devices. Using qualitative reporters can be applied in field research [70]. One good example is the detection of parasites without using cold-protected samples or complicated equipment for the detection process [86].
In the following section, we provide our final overview of the impact of E. coliin the synthetic biology future.
5. Genetic engineering and synthetic biology of E. coli
With the avenue of in vitroDNA synthesis to generate larger fragments with increased fidelity along with novel assembly methods, we are now capable of generating large and custom-made DNA molecules with the desired properties or even without the source of a DNA sample having only the sequence itself. New biological parts (genes, promoter sequence, terminators, etc.), devices (gene networks), and modules (biosynthetic pathways) are only limited by our imagination and the drive to create them. Also, without the advancement of methods for analyzing large amounts of data, bioinformatics, codon optimization software, genome mining, and user-friendly databases, synthetic biology creations are permeating in many laboratories around the world. With this in mind, we will review the current technologies for synthetic genes and genomes, and how this technology can be applied in generating novel regulatory circuits and even whole genomes. In this regard, E. coliis the key organism for such endeavors. Why? Well, in this section, we provide some examples that we consider may be helpful in the future of mankind and are in our opinion relevant in the field of genetic engineering and synthetic biology.
Synthetic biology is a relatively new branch of molecular genetics that incorporate engineering principles for modifying several aspects of cell physiology, rewiring genetic circuits, creating novel circuits, and synthesizing custom-made DNA sequences and even genomes [87]. This particular branch of biology needs to be supported by an extensive knowledge of the organism that modifications or even whole genome synthesis is attempted, several novel tools for analyzing big datasets and molecular tools for that particular organism, for the generation of sequences and the computational design of DNA molecules, and a goal that can be achieved with the desired organism. E. colialong with Saccharomyces cerevisiaeare the most studied and well-comprehended organisms in science, and diverse phenotypes have been identified that are helpful for bioengineering [88, 89].
Multiplexing is the novel approach for redesigning organisms to do desired tasks [90]. By cycling through design-build–test framework we can achieve novel features in existing proteins and can further the advancement of genetic engineering. Thus far the most complicated and time-consuming part of this framework is the testing of the novel designs. High-throughput approaches have led to the development of fast and reliable screening methods and advances in this area, such as designed biosensors for the screening of metabolite producing strains or high-throughput methods for product screening, where the use of fluorescent proteins, colorimetric assays, and mass spectrometry are cornerstones for the development of screening methods for assessing success in strain engineering [90, 91]. DNA sequencing and synthesis coupled with good screening methods is the platform for future tools for the development of designed microorganism that can do desired tasks.
With all the technologies available, the advancement of using E. colifor biotechnological applications based on synthetic approaches have led to the development of strains capable of synthesizing several novel compounds. In the following lines, we provide some examples that we find important for improving environmental conditions and human well-being.
Synthesis of important metabolites can be difficult, and researchers must face the stubbornness of microorganisms to redirect carbon flux to their own processes, rendering the production of relevant molecules costly and inefficient. But E. coliis a flexible platform for the efficient production of molecules for the pharmaceutical industry, metabolites and molecules relevant for food additives, pigments, and more recently complex aliphatic molecules [92].
Several aromatic compounds have been successfully synthesized in E. colidue to their biological activities (vitamins and antioxidants, for example), pigmentation (applied in different industrial processes), and fragrance etc. [93]. In the case of the perfume industry, the synthesis of other relevant compounds such as precursors for Ambrox, a highly appreciated odorant for the perfume industry, or the synthesis of Geraniol, a valuable acyclic monoterpene alcohol, is also used in the perfume industry and pharmaceutical applications [94, 95].
Another relevant area was E. colistepping in to biofuel production. The twentieth century is characterized by the human dependence on fossil fuels. They participate in a myriad of processes, and the demand is increasing. In order to alleviate the demand, scientists have turned to the development of novel technologies for biofuel production by the conversion of carbon sources into usable fuel. There are several reports where E. colihave been successfully engineered for the synthesis of branched-chain fatty acids or short-chain fatty acids that can ultimately lead to the mass production of fuel precursors or useful materials derived from oil [96–98]. Perhaps, the most promising future is a fully replicated fossil fuel, i.e., a mixture structurally and chemically identical to the fossil fuels that are currently under use, which is a mixture of aliphatic n- and iso-alkanes of various chain lengths [99]. Also, a more complete metabolic atlas of E. coliis needed, and recent efforts have mapped the metabolic flux from this bacterium further [100].
Another important field where E. coliis making an important contribution powered by synthetic biology is the antibiotic production. From the biomedical standing point of view, increasing antibiotic resistance in pathogens is a burden for humankind, and the discovery of novel compounds is a time-consuming task. Recent efforts with known polyketides have started to give good production rates in E. coli, favoring the process of antibiotic production from different sources, eliminating the need for host growth standardization, and inducing conditions [101].
Due to the modularity of polyketide synthases, they are excellent candidates for engineering, for either the production of novel compounds from existing gene clusters (through module shuffling, mutagenesis or deletions), or by the introduction of novel environmentally sequenced gene clusters and heterologous production [102, 103]
Therefore, the production of diverse metabolites or biological or industrially relevant molecules can be successfully achieved in E. coli. Success stories have been published more frequently. With the advancement of high-throughput technologies, more sensitive detection, and analytical tools, and better DNA synthesis, the future for E. coliis brilliant.
6. Future
With all the research that is currently conducted in E. coli, we envision that the future for this versatile microbe is bright. Gained knowledge on E. coliis overwhelming, nearly 340,000 research papers with the "Escherichia coli" keyword available in Pubmed, versus 115,000 using "Saccharomyces cerevisiae" as a keyword for an example. Several genomes from environmental sources have been sequenced. Thousands of research papers aimed to assess the metabolic and genetic potential of this organism have been published. Now we need to start joining forces for boosting the true potential of this wonder microbe.
With all the data available, we are on the verge of really important findings and novel biotechnological procedures using E. coli. Recently, with an extensive analysis of human microbiota, we are approaching exciting times where all the knowledge can be applied to help people during certain diseases related to colon microbiota imbalances [104]. Findings suggest that microbiome can be manipulated to improve certain metabolic pathways [105].
In the rest of the biotechnology fields, we have enough information that suggests E. coliwill be in the spotlight for quite some time. Even with novel organisms that have been proposed to substitute it, like Vibrio natriegens. Recently, Lee and collaborators analyzed the genome sequence and growth properties of the fast-growing bacterium V. natriegens[106], where authors created a novel platform for genetic engineering in one of the fastest growing bacteria. In previous reports, it has been shown that V. natriegensare capable of being transformed with E. coliplasmids [107], rendering that all the molecular tools available thus far may be compatible with this new system. This bacterium as E. colirenders only one important drawback: it may be pathogenic to oysters [108]. But as with old E. coli, we should be careful when managing genetically modified organisms. With recent efforts to generate lab-contained organisms, E. colimutants that can only survive in the presence of synthetic amino acids have been created [109]. Despite all the molecular tools developed so far for E. coli, the future is still open for a novel molecular toolbox. Until a novel toolbox is standardized and incorporated into the everyday life of the laboratories around the world, we will keep exploiting the capabilities of E. coli, one wonderful bug.
The authors acknowledge the generous support from CONACyT-CIBIOGEM grant CB-4O-040116, CONACyT grant CB-2012-01 182671, and Guanajuato University grant 1081/2016 (DAIP). NVM acknowledge the support from CONACyT for a scholarship grant. We also acknowledge the support from Dr. Felipe Padilla Vaca.
2.Plasmid and the E. coli revolution
3.Genome modifications to understand E. coli
4.E. coli-based biosensors: tools for many applications
5.Genetic engineering and synthetic biology of E. coli
6.Future
Vargas-Maya Naurú Idalia and Franco Bernardo (July 12th 2017). <i>Escherichia coli</i> as a Model Organism and Its Application in Biotechnology, <i>Escherichia coli</i> - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications, Amidou Samie, IntechOpen, DOI: 10.5772/67306. Available from:
10Crossref citations
Biosensor Platforms for Rapid Detection of E. coli Bacteria
By Rodica Elena Ionescu
Anopheles mosquitoesNew insights into malaria vectorsEdited by Sylvie Manguin
Anopheles mosquitoes
Edited by Sylvie Manguin
The Phylogeny and Classification of Anopheles
By Ralph E. Harbach


Life is pretty crazy, I think everyone can agree on that. I decided I needed a little time off from the blog just to focus on my own life. I have a lot of things going on at the moment and felt like I needed to devote more time to my other priorities. So for those that are wondering, here's what I've been up to.
School: I was absolutely swamped with assignments and final exams for college, as this past week was the last week of classes for the semester. I felt more than overwhelmed and definitely needed to buckle down and focus over this last week so that I can uphold the grades I've worked so hard for. The semester is finally over! And while I'm not graduating this semester (thanks to transferring schools/degrees), I'm beyond relieved to be done for the time being. This last week was rough, but I finished strong! I should be finishing this semester with a 3.9 GPA.
Birthing/labor research: As I get closer to baby's due date, I've been feeling so unprepared for what's about to happen. I found that I was letting some fear creep in and decided I need to change that. I've been taking some time each day/ evening to do some research on labor and practice what I can to prepare me for the big day! (24 days away…what.) Tim has been helpful in reminding me that these fears are not coming from me or from God and to pray for God to cast them out ("For God hath not given us the spirit of fear; but of power, and of love, and of a sound mind" – 2 Tim. 1:7, KJV). Our bodies were made to do this and our minds are capable of so much more than we think!
Family: Pretty much any of you who are college kids (or who have kids in college) know that this time of year is graduation time! My brother graduated from Grove City College over the weekend and was able to come down and see me. I was pretty upset I couldn't be there to celebrate his graduation, since it's over a 5 hour drive, but I'm so glad I was able to see him at all. It's been a little while since I've seen either him or my dad actually, so it was great that he and my parents could visit this weekend.
I've also been really feeling the need to spend as much time with Tim as I can, seeing as in just 24 days we will no longer be a couple – but a family of 3! I'm so excited, yet at the same time my heart still breaks a little to think of losing the precious alone time I get with my husband. So part of the reason I've been off the blog for a bit has been to spend more time with him. Now that the weather's getting nicer, we're planning on taking walks every evening that we can. I look forward to enjoying our last few weeks together as a "couple without kids".
Other responsibilities: Though this is one section, its probably the real reason I put the blog on the back burner for a few days. That's because we all know its the combination of things that usually overwhelm us. It's just been a little too much for me to think about recently and I didn't want to post something that wasn't genuine or heartfelt. These "other responsibilities" have included dentist visits, birthing classes (or trying to…had to reschedule 3 times), prenatal appointments, work, cooking, prepping the room for baby, looking up a pediatrician, looking for health insurance for the three of us, making calls regarding payments for the prenatal appointments, birth, and postnatal appointments, and still trying to get enough sleep to function the next day. Not to mention the fact that this past week has been probably the hardest work week I've seen my husband go through. Several of the days that I was off work, I even went with him and helped him however I could (usually just taking numbers down). Last year he broke his foot 6 weeks before our wedding and I went to work with him every day. Since I was basically a coworker for him during that time, I know a lot about what he does and how to do certain parts of it.
The last thing I'm gonna say is this: It'll probably happen again. Taking a break from the pressures of having to think up a blog post or take good photos for my blog/instagram has been essential to my well-being and I'll probably do it again every once in a while. I would encourage this to anyone who's also a blogger/instagrammer. If you're feeling overwhelmed – take a step back. You can always pick up where you left off and if your readers truly care about you and enjoy your content – they won't care! Your mental/physical health and emotional well being is always more important – especially if you're also dealing with pregnancy/the postpartum period/a newborn/ or really any other thing that puts extra stress on your mind and body. Listen to your body, give it a break, and it will thank you!
Next Post More stripes…a pregnancy thing?
You may have come across this already, but I think red raspberry leaf tea (RRLT) and eating dates in the last weeks of pregnancy really sped up both my births. I drank RRLT every day and then had some extra-concentrated in early labor. The flavor on its own is pretty meh but add some mint or hibiscus (and nettle for some extra vitamins and minerals!).


text:
What Are The Best Mood Stabilizers For Anxiety With Bipolar?
Anxiety is a natural body's response to fear and nervousness about the upcoming event. A number of factors can contribute to anxiety such as stress at work or school, personal issues, financial stress, death of loved ones, medical illness, and side effects of medications, substance abuse, and decreased supply to oxygen due to diseases or other conditions. Phobic disorders and stress disorders also lead to anxiety. People suffering from bipolar disorder suffer from severe anxiety. Mania is characterized by elevated or irritable mood, high energy levels, hyperactive, impulsive and decreased sleep whereas depression is characterized by sadness, negative feeling, hopelessness, trouble falling sleep and lack of energy. People with anxiety disorder and bipolar disorder suffer from panic attacks, excessive worries, and obsessive-compulsive disorder (OCD). These people often take high doses of drugs or alcohol to be able to concentrate on any activity or to have good sleep.
Most people often suffer from anxiety and bipolar disorder as a co-morbid condition. The doctor's first prescription will be a mood stabilizer. Many antidepressant drugs have mood stabilizing action which prevents worsening of symptoms of mood disorders and also help in overcoming anxiety in such patients.
A doctor might prescribe a mood stabilizer in such patients depending upon symptoms and the diagnosis. If you have been suffering from an anxiety disorder since a long time then the possible medications which were effective and the side-effects associated with such medications are considered. Different drugs are prescribed for mood stabilization. Each drug has its own merits and demerits:
Carbamazepine. It is a benzodiazepine class of drug useful in treating mood disorders. It can also be used in combination with lithium. It is particularly efficient in mood stabilization of patients with early-onset bipolar disorder and people who do not have a history of mood disorders. The drug is used considering the possible interactions.
Lithium. This is considered as the best mood stabilizer for people suffering from bipolar I disorder where mania episodes are predominant. Lithium is not effective in treating severe depression. Immediate effects of the medicine will not be seen. The effect on mood can be seen after 14 days and the person comes to normal terms after a period of 3 weeks. Most of the people suffering from mania episodes respond to lithium therapy and people who do not respond to lithium therapy are prescribed along with other mood stabilizers. At the start of treatment lithium levels are checked twice in a week and once the patient symptoms improve, lithium levels are monitored every 6 months. Lithium has its own side effects as it can have detrimental effects on kidneys, thyroid and heart rhythm. Doctors advise for a renal function test, thyroid test and electrocardiogram (ECG).
Lamotrigine (Lamictal). This efficiently helps in controlling mood swings in people with bipolar II disorder wherein people suffer from severe depressive episodes with hypomania, a less severe form of mania. It is an effective anti-depressant agent and hence useful in treating depression associated with bipolar disorder. The drug is basically used for treating epileptic seizures.
Valproic acid. The drug produces its action from day 14 and helps in setting the mood especially in patients suffering from mania.
Gabapentin. This drug has been found to be an effective antiepileptic drug and when used in patients with mood disorders, it has shown a positive impact. It is also useful for patients who are not responding to lithium.
A number of herbal medicines have been found to be effective in controlling anxiety symptoms such as passionflower, kava, Valerian (Valeriana officinalis), St. John's wort, Ginkgo biloba, Ashwagandha (Withania somnifera), Green tea, Chamomile (Matricaria recutita) and Lemon balm (Melissa officinalis). The inclusion of food supplements L-lysine, L-arginine, L-tyrosine, L-phenylalanine, Omega-3 fatty acids, Selenium, Magnesium, and ascorbic acid.
Benzodiazepines, Lithium and divalproex sodium are the medicines commonly prescribed as a mood stabilizer in anxiety with bipolar. Many antidepressant drugs have antianxiety property and hence a single medication can help you in setting the mood and controlling the emotions.
Common Mood Stabilizers Used For Treating Bipolar Disorder & Its Side Effects
Can Mood Stabilizers Help With Anxiety?
Do Mood Stabilizers Make You Gain Weight?
What Do Mood Stabilizers Do To The Brain?
Various Causes Of Deterioration In Mental Health
An Excellent Mental Health Leads To An Excellent Mind
Effects of Stress, Fear, Anger & Love On The Human Brain
An Excellent Mind Leads To Excellent Results
Psychosomatic Pain: Types, Causes, Symptoms, Treatment, Alternative Therapies

redpajama_set_name: RedPajamaCommonCrawl


Needlepoint herb pillow kit to make a delightful scented pillow, or the stitched canvas can be framed or used as a panel for larger cushions.
Color-printed 12 mesh canvas, backing material, a sachet of lavender.
Full instructions, Black and white chart, needle, Appleton's wool.
Design size: 10" square (approx).
Canvas size: (13.5") square (approx).


S&Ls Savings Banks Industry's pace of Revenue growth in 4 Q 2018 accelerated to 8.77 % year on year, above Industry average.
Sequentially Revenue grew by 16.53 %.
Within Financial sector 6 other industries have achieved higher Revenue growth. Revenue growth total ranking has impoved so far to 43, from total ranking in previous quarter at 102.
S&Ls Savings Banks Industry's pace of Revenue growth in 4 Q 2018 decelerated to 1.66 % year on year, below S&Ls Savings Banks Industry average.
Sequentially cumulative Revenue fell by -0.04 %.
Within Financial sector 9 other industries have achieved higher Revenue growth. Revenue growth total ranking has impoved so far to 87, from total ranking in previous quarter at 96.


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text:
Orange Goblin, ранее Our Haunted Kingdom хеви-метал группа из Великобритании, сформированная в 1995 году. В настоящий момент группа состоит из вокалиста Бена Уорда, гитариста Джо Хоара, басиста Мартина Миллэрда и барабанщика Криса Тернера.

История
Orange Goblin был сформирован в 1995 году и первоначально носил название Our Haunted Kingdom. Они выпустили семидюймовый сплит с группой Electric Wizard в 1996 на лейбле Rise Above, до того, как сменили вывеску на Orange Goblin. Первые три альбома группы исполнены в жанре стоунер/дум. Начиная с альбома Coup De Grace, вышедшего в 2002 в звуке группы появились включения панка, дум-метала и элементов других музыкальных стилей.

Первые пять релизов были изданы на лейбле Rise Above. Ритм-гитарист, Питер О'Мейли покинул группу в 2004, для того, чтобы начать карьеру художника. 16 декабря 2005 года группа отпраздновала своё десятилетие, дав концерт в клубе Underworld, в Камден-Тауне, при участии групп Scissorfight и Blood Island Raiders. Альбом 2007 года Healing Through Fire был издан на Mayan/Sanctuary Records.
3 сентября 2008 группа анонсировала подписание контракта с Candlelight Records и работу над материалом для нового альбома, с датой релиза, намеченной на сентябрь 2009 года. Он был анонсирован в мае 2009 словами: «Написание песен для нового альбома продолжается, но основа из четырёх, или пяти песен выглядит многообещающе. Пара черновых названий для новых песен звучит как Red Tide Rising и The Bishop Wolf's». Ранее группа анонсировала задержку в записи альбома в связи с непредвиденными обстоятельствами, и извинилась перед фанами, сказав, что «этого стоит дождаться».

12 месяцев спустя, в марте 2010 от лица группы было сделано заявление, что группа отказывается от обязательств по записи нового альбома, и не может сказать, когда в обозримом будущем будет выпущен новый альбом. Группа анонсирует запись нового материала в августе 2011, после упоминания на радио-шоу Ricky Gervais' XFM. Эта запись была завершена в сентябре 2011 и выпущена в феврале 2012 года под названием A Eulogy for the Damned на Candlelight Records.

В середине 2010 года бэк-каталог группы был переиздан в виде дигипака на лейбле Rise Above Records. В середине декабря группа отпраздновала свое пятнадцатилетие, ставшее кульминацией ежегодной Рождественской выставки в Лондоне, при поддержке прибывшей из Великобритании блюз-рок группы Firebird, состоящей из бывших членов групп Carcass и Spiritual Beggars, и американской дум-рок команды Solace.

Также, Orange Goblin выступил в 2011 году на фесте Maryland Deathfest в Балтиморе, штат Мэриленд, США.

Уже не новый альбом (2014)
В 2014 группа записывает следующий очередной альбом и издаёт его 6 октября (Англия, Евросоюз) и 7-го октября (США) через Candlelight Records. Альбом получил название «Back from the Abyss» и вошёл в чарт Великобритании под номером № 98.
«Back from the Abyss» — это звук Orange Goblin, жгущего во все цилиндры!" рычит фронтмен Ben Ward. «От Sabbathianовской тяжёлой нарезки в "Sabbath Hex", моторхедообразного панковского рыка на "The Devil's Whip", эпичного, мейденовского галопа на "Mystical Knives" к апокалиптическому думу "The Shadow Over Insmouth". Это лучшее из музыки, повлиявшей на нас, собранное в одном месте. Мы создали наиболее зрелый альбом группы Orange Goblin. После отличного мирового признания для "A Eulogy for the Damned", и последующих двухлетних гастролей повсюду, мы узнали много нового, чтобы продолжать творить. Мы были готовы и вошли в студию вооруженные убийственными риффами. Все они представлены на нашем новом альбоме и продолжают Orange Goblin-тренд никогда не бояться нового и постоянно двигаться вперёд. В ходе записи мы создали уникальный тяжёлый звук, лежащий где-то между думом, стоунером и классическим металлом, вдохновленный роком 70-х, блюзом, панком и всем, что между. Мы снова работали с Jamie Dodd и он проделал хорошую работу на последней записи, я даже думаю, что в этот раз он превзошёл сам себя. Добавив удивительный талант Джейми в живые выступления и процесс написания песен, мы сделали альбом, которым очень гордимся. Наш последний альбом дал группе новую жизнь и вырвал нас из небытия. Новый альбом показывает, как Orange Goblin в очередной раз выходит за установленные рамки… и "Back from the Abyss" готов всем врезать!»

Участники группы
Текущий состав
 Бен Уорд — вокал, гитара (с 1995 по настоящее время)
 Джо Хоар — гитара (с 1995 по настоящее время)
 Мартин Миллард — бас (с 1995 по настоящее время)
 Крис Тернер — ударные (с 1995 по настоящее время)

Бывшие участники
 Пит О'Мейли — гитара (1995—2004)

Сессионные участники
 Дункан Гиббс — клавиши для Frequencies from Planet Ten
 Джейсон Грэхем — клавиши для Healing Through Fire

Дискография
Студийные альбомы
 Frequencies from Planet Ten (1997)
 Time Travelling Blues (1998)
 The Big Black (2000)
 Coup de Grace (2002)
 Thieving from the House of God (2004)
 Healing Through Fire (2007)
 A Eulogy For The Damned  (2012)
 Back from the Abyss (2014) UK #98
The Wolf Bites Back (2018)

Концертные записи
 A Eulogy For The Fans (2013)

EP
 Electric Wizard/Our Haunted Kingdom (1996)
 Nuclear Guru (1997)
 Chrono.Naut / Nuclear Guru (1997)
 The Time (1998)
 Orange Goblin/Alabama Thunderpussy (2000)

Синглы
 «Some You Win, Some You Lose» (2004)

Участие в сборниках
 «Saruman's Wish» в Dark Passages II (1996)
 «Aquatic Fanatic» в Stoned Revolution — The Ultimate Trip (1998)
 «Quincy the Pig Boy» в Rise 13 (1999)
 «Black Shapes of Doom» в Bastards Will Pay: Tribute to Trouble (1999)
 «No Law» в High Volume (2004)

Примечания

Ссылки

Музыкальные коллективы, появившиеся в 1995 году
Музыкальные коллективы из Лондона
Квартеты
Хеви-метал-группы Великобритании

redpajama_set_name: RedPajamaWikipedia


text:
Cocktails and Candy Canes
Good To Go Jamaican Cuisine & Event Space, Evanston, IL
The end of the year is here and what better way to celebrate it other than with cocktails and candy canes!
**Goodie Bags will be provided**
I'm a newer real estate agent in the Chicagoland area, and I would like to get to know more potential homebuyers, investors, agents and real estate professionals.
Even if you aren't interested in homeownership, it's good to have a strong union and I'd love if you came out to me and my partners this amazing black owned spot in Evanston called Good to Go Jamaican.
ABOUT GOOD TO GO
Good To Go is a Jamaican Cuisine restaurant that fuses the mouth-watering taste of Caribbean food with
exciting new tropical cocktails and craft beer. Our house specialties include Jerk Chicken, Oxtail, Brown Ste
Chicken, Curry Goat, & Jerk Pork. Our Tropical Cocktails, imported Jamaican beverages, fresh squeezed
vegetable juices and island smoothies complete our island offerings.
Good To Go was established by husband and wife team, Tony and Lenice Levy in 2002. It started as a small
quick service restaurant located on the Chicago side of Howard Street and after 16 years of building a strong
following and dynamic business, they purchased, completely renovated and opened a new location on the
Evanston side of Howard Street in 2018.
Lenice is originally from the south side of Chicago and attended DePaul University where she received her
Baqbelors degree in Marketing. Her 20 plus years in corporate America equipped her with the skills to manage
all of the administrative, HR and corporate aspects of running a successful business. Her passion for
architecture, business development, and property development has been intricate in the design and build of
the new venue which is bursting with design elements that are unique and one of a kind
At the age of 27, Tony Levy, migrated to the United states from Jamaica. Being the eldest of 11 siblings created
many opportunities for him to prepare daily meals alongside his mother. Over the years he developed a strong
passion for cooking and was delighted seeing people enjoy the tasteful entrees he would prepare. His passion
eventually turned into a burning desire to open an establishment to cater to a broader audience. His three
most notable creations include his homemade JERK SAUCE where the recipe originates from his great
grandmother. His all-natural house blended juice titled "STAMINA DADDY. Infused with various ingredients
like oats, okra, green banana, peanuts, tonic and stout, STAMINA DADDY has become a household name for
energy and stamina amongst the male patrons. And, finally the JERK CHICKEN which is the household favorite
by all.
Dec 29, 2021 at 6:00 PM EST
Good To Go Jamaican Cuisine & Event Space
711 West Howard Street


text:
Ayhan Karakuş (ur. 13 października 1989) – turecki zapaśnik w stylu klasycznym. Olimpijczyk z Londynu 2012, gdzie zajął szesnaste miejsce w kategorii 55 kg.

Zajął piąte miejsce w mistrzostwach świata w 2009. Czwarty w Pucharze Świata w 2015 i ósmy w 2010 i 2012. Brązowy medalista igrzysk śródziemnomorskich w 2009 i akademickich MŚ w 2016, a także igrzysk solidarności islamskiej w 2021. Drugi na mistrzostwach śródziemnomorskich w 2010. Trzeci na mistrzostwach Europy juniorów w 2009 roku.

Przypisy

Bibliografia
 Ayhan Karakuş na stronie www.iat.uni-leipzig.de
 cd.

Uczestnicy Letnich Igrzysk Olimpijskich 2012
Tureccy olimpijczycy
Tureccy zapaśnicy
Urodzeni w 1989
Ludzie urodzeni w Sivasie


text:
Q: Open URL in a mobile browser from the inApp webview using Javascript Assuming that I called a webpage using a Flutter webview plugin. The webpage code are as follows :
<button onclick="window.open('https://api.whatsapp.com/send?phone=60175XXXX','_system');"> Whatsapp</button>

When I clicked on the button, it redirects to ERR_UNKNOWN_URL_SCHEME because the link pointed to whatsapp:// , not https:// .
I am trying not to adjust the Flutter code, by not following the Android WebView err_unknown_url_scheme .
I just want to use Javascript inside the webpage to open the mobile browser.
Already tried to use '_blank' or target='_system', but it doesn't work.
"window.open('https://api.whatsapp.com/send?phone=60175XXXX','_blank');"

...

Any helps? Thank you.
My question might be similar with this one, but the answer doesn't help much.
How to open a window(poup) on mobile browser from inside a webview using javascript?

redpajama_set_name: RedPajamaStackExchange


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This WordPress theme sports a very simple design. However, it can be fully modified and its colors can be changed. You can new photos to it and add descriptions to them.
It is a fully editable, completely responsive WordPress template for real estate websites. The post formats can be personalized. It only takes two step to install this template and users of this template are promised to receive lifetime updates for free.


Tuition for the 2018-2019 school year ranges from $5,020 (two mornings/week) to $21,920 (five days/week, 8 am to 5:30 pm). Download a pdf with the full list of current school fees, including extended day hours, here.
At SJP, we are committed to admitting students of diverse social, economic, and cultural backgrounds, regardless of their families' financial means. We offer tuition assistance to qualifying families, based on their demonstrated financial need and the school's available funds. Information about how to apply to our tuition assistance program will be provided upon admission to the school, and we encourage all admitted families to apply. If you would like to learn more about the program before submitting an application, please contact Roger Setterfield, School Director, at 415-752-8258.
In addition to our tuition assistance program, St. James is also proud to be a provider school in San Francisco's Preschool for All program, which allows us to offer tuition offsets to the families of our PreK students who live in San Francisco. More information about the program can be found here.
Nondiscrimination policy: St. James Preschool, a nonsectarian school, does not discriminate on the basis of race, color, sexual orientation, national and ethnic origin, and/or religious background in the administration of our educational policies, admissions policies, financial assistance, and school-administered programs.


text:
Q: About the Perl range operator I have a question about the range operator in Perl. The code is here:
 #! /usr/bin/perl
 open FILE, "<test.txt";
 while (<FILE>) {
     print if (1 .. 5);
 }
 close FILE;

The contents in test.txt is something like:
 1
 2
 3
 4
 5

The result of the code is all five lines of the file. There is no difference when I use print if (1 ... 5). I think it should output four lines.
Is there something I made a mistake in?

A: The contents of the file actually don't matter here. What does matter, however, is the line number (of each processed line), as that's what gets checked against each operand of the .. operator. It's actually can be written as...
print if ($. == 1) .. ($. == 5);

By the rules of flip-flopping, print will start to work when line number becomes equal to 1, and finishes when line number becomes equal to 5. The only difference between .. and ... in flip-flop is about testing both operands on the same expression, which is not relevant in this case.
What you might have wanted to use was ...
print if $_ ~~ [1..5];

... that is, testing the contents of the line against a range. But there is no difference between .. and ... in a list context - they both return the same range:
print for (1..5); # 12345
print for (1...5); # 12345 as well

And now for something... well, not so completely different. ) Consider the following:
while (<DATA>) {
  print '..', $_  if 1..($. >= 1);
  print '...', $_ if 1...($. >= 1);
}
__DATA__
1
2
3

It prints...
..1
...1
...2

... and then nothing, because...

*

*The .. operator checks both conditions on the same - first - line. Checking the first condition ($. == 1) switches it on (and makes the result of the whole .. expression equal to 1). But guess what? It's immediately switched off then, as $. >= 1 also evaluates to 1 (true). As flip-flop is switched off, all the next lines have to pass the first condition again, and that just can't happen.

*The ... operator is more relaxed. For the first line, as the first condition is truthy, the gateway is opened - but the second expression is not checked here! It will be checked, though, for the second line, closing the gateway - yet 2 manages to get through.

A: In your case it doesn't matter as .. can't do flip-flop for the same line number.
To quote perlop:

In scalar context, ".." returns a
  boolean value. The operator is
  bistable, like a flip-flop, and
  emulates the line-range (comma)
  operator of sed, awk, and various
  editors. Each ".." operator maintains
  its own boolean state, even across
  calls to a subroutine that contains
  it. It is false as long as its left
  operand is false. Once the left
  operand is true, the range operator
  stays true until the right operand is
  true, AFTER which the range operator
  becomes false again. It doesn't become
  false till the next time the range
  operator is evaluated. It can test the
  right operand and become false on the
  same evaluation it became true (as in
  awk), but it still returns true once.
  If you don't want it to test the right
  operand until the next evaluation, as
  in sed, just use three dots ("...")
  instead of two. In all other regards,
  "..." behaves just like ".." does.
The right operand is not evaluated
  while the operator is in the "false"
  state, and the left operand is not
  evaluated while the operator is in the
  "true" state. The precedence is a
  little lower than || and &&. The value
  returned is either the empty string
  for false, or a sequence number
  (beginning with 1) for true. The
  sequence number is reset for each
  range encountered. The final sequence
  number in a range has the string "E0"
  appended to it, which doesn't affect
  its numeric value, but gives you
  something to search for if you want to
  exclude the endpoint. You can exclude
  the beginning point by waiting for the
  sequence number to be greater than 1.
If either operand of scalar ".." is a
  constant expression, that operand is
  considered true if it is equal (==)
  to the current input line number (the
  $. variable).

A: Thank you for your answers which enlighten me. After reading perldoc and doing some tests, I want to share my understanding here. If there is something wrong, please let me know. :)
print if (1 .. 5) is a shortcut for print if ($. == 1 .. $. == 5). The initial state of the expression is false, after reading the first line, $. == 1 is satisfied and the state turns to true. So the first line is printed.
Then the second line, the third, the fourth.
When it comes to the fifth line, the state is still true. This is the state that decides whether to go into block. So the fifth line is also printed, then $. == 5 is satisfied, at this time, the next state becomes false. So if there were any line afterwards, they will not be printed.
There is no difference between .. and ... here, because the difference only takes place when the left operand returns true. Let me show it:
The example here is select from perlop. This example confuses me at first, but let me show my understanding.
 @lines = ("   - Foo",
           "01 - Bar",
           "1  - Baz",
           "   - Quux");
 foreach (@lines) {
     if (/0/ .. /1/) {
         print "$_\n";
     }
 }

When it comes to "01 - Bar", /0/ is satisfied, so the state becomes true. And at the same time, /1/ is also satisfied, so the next state suddenly becomes false, this is where .. is different from ..., because ... will not evaluate /1/ now, and the next state will still be true. So "1 - Baz" will not be printed out. Otherwise, when ... is used, this line can be printed out.

A: In Perl, something is false if it returns either an undef, a null string, or a 0 value. Otherwise, it is considered true.
You're saying:
print if (1..5);

This means the contents of $_ will print out if (1..5) returns a true value. Well, (1..5) returns an array of five members. That means if (1..5) is always true, and each line in your file will print.


The Yankees have signed Mike Zagurski to a minor league deal, MLBTR's Tim Dierkes has learned. His contract contains an August 15 opt-out date if Zagurski has not been promoted to the Majors.
Zagurski, 30, elected free agency on Wednesday after being designated for assignment by the Pirates last week. While the left-hander made just six big league appearances this season for the Bucs, he has been extremely sharp in Triple-A, posting a 2.14 ERA with 37 strikeouts and nine walks across 21 innings of work.


text:
Air Canada Stock: What Could It Go In 2023?
Air Canada (TSX:AC) stock has started 2023 on a strong note, as it jumped by 4.2% in the first week of the year, closing above the key psychological level of $20 per share for the first time since November 1. With this, AC stock reached its highest level in over six months. By comparison, the TSX Composite Index also rose by 2.2% last week. Let's take a closer look at key fundamental factors that drove it higher last week before discussing how high Air Canada stock could go in 2023.
Why Air Canada stock jumped last week
Last week's sharp gains in Air Canada stock could partly be attributed to the renewed buying in Canadian growth stocks at the start of the year — especially in most beaten-down stocks. This renewed buying was mainly underpinned by investors' hopes that the stock market performance could improve on a YoY (year-over-year) basis in 2023.
In addition, in the week ended on January 6, Statistics Canada revealed that domestic employment jumped by 104,000 in December with the help of a sharp gain in employment among youth between 15 to 24 years of age. This employment report was significantly stronger than Street's expectations, with the second highest job additions in a single month since March 2022, boosting investors' confidence and triggering a market-wide rally, including in Air Canada's share prices.
But challenges might not be over yet
In the quarter ended in September 2022, Air Canada turned profitable for the first time since the start of the pandemic. During the quarter, its total revenue jumped by 153% YoY to $5.3 billion with continued strong travel demand amid easing COVID-related restrictions. This helped the Canadian flag carrier post $1.23 per share in adjusted quarterly earnings, massively better than Bay Street analysts' estimate of $0.43 per share.
However, it might be difficult for Air Canada to remain profitable in the next few quarters as high costs due to YoY increase in fuel prices along with higher wages, salaries, and benefits for its employees. The surging costs could be one of the key reasons why analysts don't expect the airline company to report net losses in the first and second quarters of 2023.
So, how high could AC stock go in 2023?
Overall, Air Canada stock seems to have started the new year on a strong note by posting more than 4% gains in the first week itself. But that doesn't mean that the economic uncertainties from 2022 have suddenly disappeared this year.
In fact, many economic experts and large banks last year predicted that Canada and the United States might enter a moderate recessionary period in early 2023. While market bulls managed to close the first week of 2023 on a positive note, Air Canada stock could still be extremely sensitive to any new macroeconomic developments, which can keep it highly volatile in the near term.
Air Canada currently has a market cap of $7.3 billion, as AC stock trades at $20.20 per share, down 58% from its pre-pandemic year 2019's closing price of $48.51 per share. Despite its continued progress on the path to financial recovery, these big losses make it look undervalued to buy for the long term. Given macroeconomic uncertainties, it's nearly impossible for anyone to predict how high Air Canada stock could go in 2023. But keeping its strong underlying fundamentals in mind, it could be an attractive bet for long-term investors at the current price despite expected short-term volatility.
The post Air Canada Stock: How High Could it Go in 2023? appeared first on The Motley Fool Canada.
Should You Invest $1,000 In Air Canada?
Before you consider Air Canada, you'll want to hear this.
Our market-beating analyst team just revealed what they believe are the 5 best stocks for investors to buy in January 2023… and Air Canada wasn't on the list.
2 Airline Stocks That Could Be Clear for Lift Off
Why Air Canada Stock Fell 8% in 2022
2 Canadian Stocks That Could Course-correct in 2023
Does Air Canada Stock Have What it Takes to Double to $40?
Canadians: 2 Air Travel Plays to Watch in 2023
The Motley Fool has no position in any of the stocks mentioned. The Motley Fool has a disclosure policy. Fool contributor Jitendra Parashar has no position in any of the stocks mentioned.
By: Jitendra Parashar
Title: Air Canada Stock: How High Could it Go in 2023?
Sourced From: www.fool.ca/2023/01/09/air-canada-stock-how-high-could-it-go-in-2023/
Published Date: Mon, 09 Jan 2023 19:30:00 +0000
aircanadathefirst


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be immersed in a rich, English speaking environment to build their understanding, confidence and enjoyment of the language.
This course is a great opportunity for your child to develop their English language proficiency. The carefully selected scripts give students essential practice in oral reading fluency and public speaking. The scripts also provide an opportunity for group interaction and student cooperation.


Thingvalla brochure page 50 This booklet was issued by the Thingvalla Line in 1887, and gives an interesting insight in the progress of emigration. This is a promotional pamphlet which was forwarded to potential travelers and emigrants. The booklet is written in Danish and Swedish.
Description : This booklet was issued by the Thingvalla Line in 1887, and gives an interesting insight in the progress of emigration. This is a promotional pamphlet which was forwarded to potential travelers and emigrants. The booklet is written in Danish and Swedish.


text:
Cody Weddle, Colombia/Venezuela Correspondent
Andrea Torres, Digital Reporter/Producer
Tags: Colombia, Travel
Feds warn Americans to avoid all travel to Bahamas
Colombia reaches 1 million confirmed coronavirus cases
Thousands want out of hurricane-ravaged Colombian island
ISLA DE LA PROVIDENCIA, Colombia – Thousands of homeless Colombians are trying to find their way out of a remote hurricane-ravaged island off the coast of Nicaragua. Eight days ago, Category 5 Hurricane Iota destroyed Providencia, a mountainous Colombian island in the Caribbean.
Virginia Webster, who is among the island's natives who speak English-based creole, belongs to the Miskito ethnic group. She has survived hurricanes before, but this one, she said was different.
"It was like you lit a fire and it passed through the whole island because it got everything," Webster said.
The island's residents have lost their jobs. The small tourism industry — which was carefully built around the emerald-green nature reserves, pristine golden beaches, and UNESCO-protected coral banks — is merely a shadow of what it used to be before the coronavirus pandemic and the catastrophic Nov. 16 hurricane.
Rastafarians' reggae parties at the paradisiac island with an English-Dutch-French-African cultural influence have come to an end. The locals' acceptance of their losses is arriving in stages. Some of the residents appear to still be in shock as they continue to assess the damage and wait for government aid.
Laidy Betancourt said she, her husband, and their 10-year-old son lost everything. She injured her leg amid the chaos of the storm. With nearly all of the island's infrastructure destroyed, Betancourt and dozens of others sought refuge in what is left of the Our Lady of Sorrows, a small Roman Catholic church.
Rev. Benito Huffington, the parish's priest for more than three decades, said he and three nuns waited inside a windowless restroom. He said there were three stages: The storm started to beat the island about 8 p.m. The eye of the storm was on them about 3 a.m. The final hit was about 10 a.m. the next day. There were 33 people inside the church's youth center when the roof collapsed.
"That was what finished everything off," Huffington said,
While they find their way out of the island, the Betancourt family is among the dozens who have found refuge at the church. They are keeping a few belongings inside one of the 1,500 government-issued tents, and they are all sleeping on the floor. Authorities are discouraging travel during the coronavirus pandemic, but there have been some evacuation flights.
Felipe Cabezas, a well-known scuba diving instructor known as "Fepo," owns the island's Felipe Diving Center. The brave man who isn't afraid to take tourists shark diving was in tears as he remembered how held tight to a plastic bathroom door. He survived in the windowless room with his wife, sister and nephew.
"The only thing we did was pray ... everything was shaking, and this was all going to fall," Cabezas said. "One more hour of that and we would be dead."
Providencia Mayor Norberto Gari Hooker has been asking the Colombian government in Bogotá for help since identifying Rogino Livingston, 47, as the only resident who died during the storm.
This week rescue workers were still finding dogs that had been trapped in the rubble. Colombian authorities set up a temporary hospital with limited resources on the island of some 5,000 residents. And to help them stay connected, officials are setting up WiFi zones while the networks are repaired.
Cabezas owns a working generator and he is using it to help hundreds of people to charge their mobile phones.
The Colombian government released this picture of Providencia, Colombia after Hurricane Iota on Nov. 17, 2020. (Efran Herrera / Oficina de prensa de la presidencia de Colombia via The Associated Press).
Colombian President Ivan Duque promised humanitarian aid and tasked a Colombian Navy team to assist with the Herculean task of rebuilding the island. Officials hope to complete the construction of about 1,490 homes in two years.
Hurricane Iota, the 30th named storm of this historic Atlantic hurricane season, also left death and devastation in Nicaragua and Honduras, which were still recovering from Hurricane Eta. The hurricane season ends on Nov. 30.
Local 10 News' Andrea Torres contributed to this report from Miami.
Andrea Torres
The Emmy Award-winning journalist joined the Local 10 News team in 2013. She wrote for the Miami Herald for more than 9 years and won a Green Eyeshade Award.


text:
http://tvtropes.org/pmwiki/pmwiki.php/Trivia/SpyroTheDragon
Trivia / Spyro the Dragon
FanficRecs
More Less - More - Haiku Narm WhatCouldHaveB…
Create New - Create New - Analysis ImageLinks Laconic PlayingWith Quotes Recap ReferencedBy Synopsis Timeline
Spyro: Year of the Dragon
Spyro: Enter the Dragonfly
Spyro: A Hero's Tail
Spyro: Shadow Legacy
The Legend of Spyro
Bad Export for You: The Japanese release of the first two games was plagued by numerous issues, including numerous glitches, a nearly uncontrollable camera, and most inexplicably, signs scattered literally everywhere that caused "helpful" tips to pop up whenever they were hit with Spyro's flame, accidentally or otherwise. Understandably, the first two games did not do well in Japan, and as a result Year of the Dragon never made it there.
Channel Hop: Went from Universal Interactive for the first few games, then to Vivendi Universal (the name they assumed before the NBC merger, after having merged with a French conglomerate), who then organized Spyro under video game stalwart Sierra, who they had bought a few years before. And after that (after most of Universal had become part of GE, then Comcast), the remains of VU/Sierra went to Activision.
Executive Meddling: Heck, this whole franchise was built around this trope. Originally, Sony of America wanted to create a "kid friendly game and character for the PlayStation" (considering the games at the time were... rather adult and teen oriented), hence the creation of the franchise, in which Insomniac never even had the full rights of in the first place, which resulted after Year of the Dragon's release that the franchise was sold and then became a Franchise Zombie until purchased by Sierra, who being way past their time ended up being bought out by Activision along with the rest of the Universal/Vivendi group.
Follow the Leader: The original trilogy was obviously made to ride the trend of collect-a-thon platformers started by the success of Super Mario 64.
Hey, It's That Sound!: For whatever reason, the sound heard when you light a tiki statue in Season of Flame's Tiki Torches world is the sound used in the Crash Bandicoot games when Aku Aku is equipped.
Image Source: For a fair amount of pages:
Spyro the Dragon (1998): Belly Flop Crushing, DemonicSpiders.Platformers, The Overworld, UglyCute.Video Games
Spyro 2: Ripto's Rage!: Everything's Louder with Bagpipes
Spyro: Year of the Dragon: Ancient Grome, Crystal Landscape, Slippy-Slidey Ice World
Spyro: Enter the Dragonfly: Wing Shield (also the Trope Namer)
Spyro: A Hero's Tail: Portmanteau (lower picture), Whole Costume Reference (right half)
Spyro Reignited Trilogy: Cowardly Mooks, Flying Flightless Bird, Moe.Video Games, Rabbit Magician, Ride the Rainbow, Green Hill Zone (Concept Art), Scary Jack-in-the-Box (Concept Art), Shifting Sand Land
I Want You to Meet an Old Friend of Mine: Gregg Berger and Neil Ross from Year of the Dragon had previously worked together on The Transformers (Berger was Grimlock and Skyfire, Ross was Slag and Springer) and in Spider-Man: The Animated Series (Berger was Mysterio, Ross played the Green Goblin).
No Export for You: As mentioned, only the first two games were released in Japan. Ironically, Spyro's rival/sister series was a phenomenal hit.
The Other Darrin:
In the first game Spyro was voiced by Carlos Alazraqui, then by Tom Kenny for the rest of the PSOne era plus Enter the Dragonfly, and Jess Harnell for A Hero's Tail and Shadow Legacy; Elijah Wood took the role for the Legend games, followed by Josh Keaton and then Matthew Mercer for Skylanders and most recently Justin Long in Skylanders Academy.
Within The Legend of Spyro trilogy, Sparx has changed voices in every game (David Spade, Billy West, and Wayne Brady), and Cynder goes from being Cree Summer to Mae Whitman, then Christina Ricci; Tobie LaSalandra rook over for Skylanders, and Felicia Day is her voice in Skylanders Academy.
All of the Spyro titles featured at least one cast change, with the exception of Enter The Dragonfly which kept all of it's characters' voice actors from Year of the Dragon. All of the cast were replaced in A Hero's Tail except for Andre Soqliuzzo (though he voices Sparx completely differently anyway).
Elora and Handel's actress has changed every time the two have appeared in the same game: Melissa Disney in Ripto's Rage!, Carolyn Lawrence in Year of the Dragon and Cassandra Lee in the Reignited Trilogy.
Refitted for Sequel: Ember was originally intended to appear in Spyro: Enter the Dragonfly, but her planned appearance was cut as a result of the game being Christmas Rushed. She would later make her official debut in Spyro: A Hero's Tail.
Talking to Himself:
Spyro and Sgt. Byrd have been voiced by the same actor in both games Bryd's been voiced in: Tom Kenny in Year of the Dragon and Jess Harnell in A Hero's Tail. Considering Spyro has to interact with Byrd to start the latter's stages, a good part of Byrd's dialogue is spent talking to Spyro.
Ripto and his minions Crush and Gulp are all voiced by Gregg Berger, who also voiced Hunter before Robbie Daymond took over the role from the Spyro Reignited Trilogy onwards.
Vaporware:
Prior to Enter the Dragonfly, there was at one point a Spyro game in the works for the Xbox.
Two separate spinoff games based off Agent 9 were in development. The first, Prime 8, was loosely based on the original character while the second game, Agent 9, was more true-to-text.
The Wiki Rule: The Spyro Wiki .
« Trivia »


Yeah, I will definitely use a sound.
Out of curiosity could you add a password to remove the new hud. I personally enjoyed the mystery of leveling up in the original game and considering as far as huds go are you gonna make reds life and power bloody like in the pasta?
(10-29-2017, 06:55 PM)Retarted Wrote: Out of curiosity could you add a password to remove the new hud. I personally enjoyed the mystery of leveling up in the original game and considering as far as huds go are you gonna make reds life and power bloody like in the pasta?
I could add an option to the main menu to use the old HUD (both Health/Power bars red and no level indicator).
As for Red's Health/Power bars, I haven't decided yet, but since it's the main villain of the game, I think it could receive some special treatment.
I definitely think it's an improvement over the original NES game. Honestly, there were so many times where I thought I was going to die, only to realize it was just the power meter. I like that you made an actual leveling system, that's really nice too.


Weddings come with a huge price tag. Between wedding essentials and wedding luxuries, the modern bride will try to DIY anything she can! Here are our top DIY wedding favors your guests (and your wallet) will love!
We love the popular Pinterest "Mint-to-be" Pun gift that has popped up all over the internet recently! This inexpensive idea is a great way to add a little humor to your wedding gifts and get guests in the fun wedding mood!
Succulents have risen in popularity over the past year, becoming the go-to wedding favor and table markers of the Pinterest world— and we love it! I recently went to a wedding where the guest favors and table markers were succulents and they were so adorable!
These are a great way to add a little humor into your wedding favors! We love the little bundles of Aspirin and mini-water bottles as a quick DIY favor for your guests. You can include a snack or two if your budget allows it — and most of these items can be bought in bulk for extra savings!
Custom name coasters are more time consuming than some of the other items on this list, but are so thoughtful! With each guest getting their own coaster, these can double as cute and custom seating cards. Ideal for the bride with a small to medium guest list, this is a perfect way to make your guests feel like they are a part of your special day!
Confetti favors are creative ways to get your guests involved in your wedding exit. You can use these as table markers and make an announcement or print out a custom tag with instructions on when to use them.
We love this idea! This is a relatively inexpensive way to give your guests a wedding favor they can actually use during your wedding— and after! Having a themed wedding? Use this favor as an opportunity to match the dancing shoes to your theme!
Midnight snack packages are the perfect way to give your guests a little pick-up after they've been dancing for hours! You can customize your snack box with whatever your guests like and even match the box to your wedding theme!
A newer idea we absolutely love, couples are switching out the traditional wedding favors for charity donations instead! The couple chooses the charity of their choice and donates the money they would spend on wedding favors to a charity instead.
Have a song you and your partner love? A song that reminds you both of your first date or when you first met? Share them with your wedding guests! A wedding CD is a cute way to get your guests involved in your love story.
Have any ideas you've tried for your wedding? Tweet them to us @revweddingfilms!


Fanfare. We're drawn. It captivates millions. Is fanfare the basis of our faith? Journey with Jesus beyond the fanfare of Palm Sunday and into His Passion.
Watch this week's Live IT Episode.
This week take the step. Receive the Gift of God more fully alive in your marriage, family and parish. Commit time with your family to talk and pray. Go here.
"How we build the Kingdom"